World Health Organization (WHO) grade II low-grade gliomas (LGGs) are slow-growing primary brain tumors, which tend to occur in young adults at their prime time of life. A majority of these patients eventually have tumor progression as aggressive high-grade glioma (HGG), and most patients eventually succumb to the disease. Immunotherapeutic approaches, such as vaccines, may be particularly appropriate. Indeed, we safely induced a robust T-cell response in patients with high-risk LGG following immunization with peptide-based vaccines targeting glioma-associated antigens (GAAs) expressed at higher levels in HGG than in LGG (NCT00795457 and NCT00874861). These studies are aimed at inducing a protective immune response in LGG patients to prevent progression to HGG. However, further refinement will require better characterization of vaccine-targetable antigens in gliomas that are progressing to HGG. We will evaluate our hypothesis that progressing gliomas demonstrate evolution in the expression profile of vaccine-targetable GAAs. Specifically, we will pursue the following two specific aims. Aim 1: Characterize the expression of vaccine-targetable GAAs in gliomas with recurrence and/or progression. Utilizing available paired gliomas from LGG patients who received multiple surgeries for recurrence and/or progression, we will evaluate changes in the expression of GAAs, from which tumor-associated peptides (TUMAPs) were derived from, in recurrence and progression. We will utilize both RNA-seq and immunohistochemistry. Our goal is to extend this line of characterization for available approximately 80 GAA TUMAPs. These studies will guide us to select most proper TUMAPs for vaccinations in LGG patients. Aim 2: Determine whether the evolution of expression profile is linked with the activation of malignancy-driving pathways (e.g., AKT-mTOR) through analyses of HLA-bound antigens. While Aim 1 studies will evaluate archived tissues for expression of available GAAs, in Aim 2, we will identify novel GAA-epitope peptides (i.e. TUMAPs) by performing human leukocyte antigen (HLA)-peptidomic-analyses and T-cell assays, targeting molecules that are expected to be up-regulated in recurrent cases (due to the activation of oncogenic pathways) and molecules that are expressed at high levels in LGG cases that recur as HGG. We hypothesize that activation of oncogenic pathways leads to HLA-presentation of novel GAA-TUMAPs that are up-regulated in the tumor cell as the result of the pathway-activation. Thus, targeting antigens that are directly relevant to the malignant transformation of LGG to HGG could improve the efficacy of immunotherapy. Impact. This research will inform the development of molecularly defined vaccines for LGG aimed at preventing their progression and malignant transformation to HGG. We are uniquely qualified to pursue the proposed study based on our extensive experience and availability of large numbers of LGG cases.